A comprehensive and validated molecular taxonomy of beaked whales, family Ziphiidae

DNA sequences from orthologous loci can provide universal characters for taxonomic identification. Molecular taxonomy is of particular value for groups in which distinctive morphological features are difficult to observe or compare. To assist in species identification for the little known family Ziphiidae (beaked whales), we compiled a reference database of mitochondrial DNA (mtDNA) control region (437 bp) and cytochrome b (384 bp) sequences for all 21 described species in this group. This mtDNA database is complemented by a nuclear database of actin intron sequences (925 bp) for 17 of the 21 species. All reference sequences were derived from specimens validated by diagnostic skeletal material or other documentation, and included four holotypes. Phylogenetic analyses of mtDNA sequences confirmed the genetic distinctiveness of all beaked whale species currently recognized. Both mitochondrial loci were well suited for species identification, with reference sequences for all known ziphiids forming robust species-specific clades in phylogenetic reconstructions. The majority of species were also distinguished by nuclear alleles. Phylogenetic comparison of sequence data from "test" specimens to these reference databases resulted in three major taxonomic discoveries involving animals previously misclassified from morphology. Based on our experience with this family and the order Cetacea as a whole, we suggest that a molecular taxonomy should consider the following components: comprehensiveness, validation, locus sensitivity, genetic distinctiveness and exclusivity, concordance, and universal accessibility and curation.     

Rapid identification of unknown heteroplasmic mutations across the entire human mitochondrial genome with mismatch-specific Surveyor Nuclease

Mitochondrial DNA (mtDNA) mutations are responsible for mitochondrial diseases in numerous patients. But, until now, no rapid method was available for the identification of unknown deleterious point mutations. Here, we describe a new strategy for the rapid identification of heteroplasmic mtDNA mutations using mismatch-specific Surveyor Nuclease. This protocol involves the following three steps: (i) PCR amplification of the entire human mitochondrial genome in 17 overlapping fragments; (ii) localization of mtDNA mismatch(es) after digestion of the 17 amplicons by Surveyor Nuclease; and (iii) identification of the mutation by sequencing the region containing the mismatch. This Surveyor Nuclease-based strategy allows a systematic screening of the entire mtDNA to identify a mutation within 2 days. It represents an important diagnostic approach for mitochondrial diseases that can be routinely used in molecular diagnostic laboratories.     

Implications of Hybridization,NUMTs, and Overlooked Diversity for DNA Barcoding of Eurasian Ground Squirrels

The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’.     

Identification of net‐winged beetle larvae (Coleoptera: Lycidae) using three mtDNA fragments: a comparison of their utility

Abstract We investigated the effectiveness of short mitochondrial DNA fragments for the identification of lycid larvae. The rrnL, cox1 and nad5 mtDNA sequences from 17 specimens of immature stages of Lycidae and Lampyridae were combined with a previously published dataset of homologous fragments representing all major lineages of Lycidae and outgroups. Their relationships were analysed under parsimony criteria. We demonstrate that high‐density profiles are necessary for accurate identification of unknown samples to generic and tribal levels and that a multilocus approach is critical for obtaining reliable results. Although widely used, the cox1 mtDNA fragment showed the worst performance for identification at genus level when the query species was not present in the library. Stronger support for deeper branches came from rrnL mtDNA. The neotenic female larvae and male adult stages of Platerodrilus sp. and Macrolibnetis depressus Pic, 1938 were associated by mtDNA fragments. Based on the present identification, larvae of Dictyopterini (Dictyopterini gen. sp., Dictyoptera aurora Herbst, 1784), Sulabanus sp., Leptotrichalus sp. (Metriorrhynchini) and Macrolibnetis depressus Pic, 1938 (Platerodrilini) are described for the first time. Further species of Platycis Thomson, 1859, Plateros Bourgeois, 1979, Macrolycus Waterhouse, 1878, Cautires Waterhouse, 1879 and Lyponia Waterhouse, 1878 are identified by morphology and molecular markers. The data on larval morphology and their usefulness for classification are discussed.     

Genetic identification of endangered North African ungulates using noninvasive sampling

North African ungulates include several threatened and emblematic species, yet are poorly studied mainly due to their remoteness and elusiveness. Noninvasive sampling provides a useful approach to obtain ecological and genetic information essential to guide conservation actions. The very first and most important step in conservation planning is to accurately identify species, and molecular genetics has been proved to be a useful tool. Several molecular genetics protocols are available for species identification, even for samples with poor quality DNA, such as faeces, hairs or bones. Most of these protocols use mitochondrial DNA for barcoding despite this marker being especially prone to problems, including mtDNA introgression, nuclear insert copies, high intraspecific diversity or heteroplasmy. In this work, we developed a molecular method based on polymorphisms in small fragments of the mitochondrial cytochrome b (cytb, mtDNA) and the nuclear kappa casein genes (KCAS, nDNA) for identifying endangered North African ungulates. These fragments revealed polymorphisms, including species‐specific variation, which allowed species identification of nine ungulate species that co‐occur in North Africa. The method was validated across more than 400 samples, including different types of noninvasive samples collected in the field. The simplicity, high reliability and relative low cost of the described method make it a promising tool to improve ecological studies of the North African ungulates and consequently, the implementation of more efficient management and conservation plans for these endangered ungulates.     

Cloning and characterization of pejerrey mitochondrial DNA and its application for RFLP analysis

Probes were cloned, characterized, and developed for all regions of the mitochondrial DNA (mtDNA) of pejerrey Odontesthes bonariensis to provide the basis for the study of genetic diversity of South American atherinopsinii and to enable species identification from small amounts of tissue. The mtDNA was extracted from liver and cleaved with Eco RI, producing four fragments (7.4, 3.4, 3.1 and 2.9 kb) which were cloned using pUC118 plasmid vectors. Sequence analysis from both ends of the fragments showed that they encode tRNA (Asp, Phe, and Ser-TGA), 12 S rRNA, cytochrome oxidase (CO) II, NADH 4, 5, and 6, and the D-loop, and that the relative positions of these genes are identical to those in the mtDNA of other teleosts. A comparison of homology with carp mtDNA nucleotide sequences revealed that tRNA (Phe and Ser-TGA) and CO II were relatively conserved, whereas the D-loop region was highly divergent. The cloned mtDNA probes detected mtDNA fragments from about 800 ng of total DNA extracted from liver, muscle, and single embryos of O. bonariensis , and were effective for restriction length fragment polymorphism (RFLP) analysis of Patagonina hatcheri , the most distant atherinopsine relative of pejerrey. The cloned mtDNA probes may be useful for the analysis of genetic diversity and non-destructive species identification, including the examination of eggs, larvae and juveniles. The mtDNA sequences reported here provide the basis for the design of primers for PCR-based RFLP analysis.     

Identification of palearctic coregonid fish species using mtDNA and allozyme genetic markers

Two types of molecular genetic markers were used for genetic identification of species and local stocks of palearctic coregonids (Coregonidae, Salmoniformes, Teleostei). Seven nominate species of whitefishes and ciscoes Coregonus , spp. of Eurasia Arctic Sea basin and inconnu Stenodus leucichthys nelma , represented by specimens from North America were studied. Using restriction analysis of PCR-amplified products of the ND-1 gene of mitochondrial DNA (mtDNA) and allelic composition at several allozyme loci discrimination was successful between C. lavaretus pidschian , Siberian whitefish, C. nasus , broad whitefish, C. autumnalis , Arctic cisco, C. migratorius , Baikal omul, C. peled , peled, and C. sardinella , least cisco. Muksun C. muksun , was indistinguishable from Siberian whitefish. Creatine kinase (CK) isozyme patterns and Rsa , I restriction patterns of ND-1 were the most effective markers allowing discrimination among species. Intra-specific differentiation in mtDNA was found in all species but was much less pronounced than inter-species variation. In several specimens composite haplotypes typical of another species were found that reflect probable gene introgression by hybridization. A combination of mtDNA and nuclear genetic markers is suggested for reliable identification of both typical species representatives and hybrids.     

Assessment of concordance among genealogical reconstructions from various mtDNA segments in three species of Pacific salmon (genus Oncorhynchus)

Seven segments of mitochondrial DNA (mtDNA), comprising 97% of the mitochondrial genome, were amplified by polymerase chain reaction (PCR) and examined for restriction site variation using 13 restriction endonucleases in three species of Pacific salmon: pink (Oncorhynchus gorbuscha), chum (O. keta) and sockeye (O. nerka) salmon. The distribution of variability across the seven mtDNA segments differed substantially among species. Little similarity in the distribution of variable restriction sites was found even between the mitochondrial genomes of the even- and odd-year broodlines of pink salmon. Significantly different levels of nucleotide diversity were detected among three groups of genes: six NADH-dehydrogenase genes had the highest; two rRNA genes had the lowest; and a group that included genes for ATPase and cytochrome oxidase subunits, the cytochrome b gene, and the control region had intermediate levels of nucleotide diversity. Genealogies of mtDNA haplotypes were reconstructed for each species, based on the variation in all mtDNA segments. The contributions of variation within different segments to resolution of the genealogical trees were compared within each species. With the exception of sockeye salmon, restriction site data from different genome segments tended to produce rather different trees (and hence rather different genealogies). In the majority of cases, genealogical information in different segments of mitochondrial genome was additive rather than congruent. This finding has a relevance to phylogeographic studies of other organisms and emphasizes the importance of not relying on a limited segment of the mtDNA genome to derive a phylogeographic structure.     

Obtaining molecular data for all life stages of Typhlodromus (Typhlodromus) exhilaratus (Mesostigmata: Phytoseiidae): consequences for species identification

Several species of the family Phytoseiidae are known to control mite pests in many crops worldwide. However, biological control success greatly depends on the accurate identification of these predatory mites. Species diagnostics is essentially based on the morphological characters of females. Thus, when only immature stages and/or males are collected, their identification is poorly supported. Molecular tools could be of great help to overcome these difficulties, as molecular sequences are assumed to be identical for the life stage considered. However, one of the essential points is to extract a sufficient DNA amount from a single specimen of immature stages (eggs, protonymphs, deutonymphs) and males (less than 300 μm in length) to amplify and sequence DNA. The markers used were two mitochondrial DNA fragments (12S rRNA and Cytb mtDNA) and the species studied were Typhlodromus (Typhlodromus) exhilaratus and T. (T.) phialatus, two cryptic species, reported to control mite pests in crops of southern Europe and commonly found on the same plants. Despite a low quantity of DNA extracted, particularly for the egg, larva and protonymph stages, DNA was amplified and sequences were obtained from all the life stages considered with the two mtDNA fragments. Sequences from all the developmental stages of T. (T.) exhilaratus were identical and well differentiated from those of its sister-species. However, contaminations were observed especially for eggs and DNA amplified with the Cytb mt marker. Utility of the present results are discussed and protocol improvements are proposed.     

The power and perils of 'molecular taxonomy': a case study of eyeless and endangered Cicurina (Araneae: Dictynidae) from Texas caves

Rapid development in karst-rich regions of the US state of Texas has prompted the listing of four Cicurina species (Araneae, Dictynidae) as US Federally Endangered. A major constraint in the management of these taxa is the extreme rarity of adult specimens, which are required for accurate species identification. We report a first attempt at using mitochondrial DNA (mtDNA) sequences to accurately identify immature Cicurina specimens. This identification is founded on a phylogenetic framework that is anchored by identified adult and/or topotypic specimens. Analysis of approximately 1 kb of cytochrome oxidase subunit I (CO1) mtDNA data for over 100 samples results in a phylogenetic tree that includes a large number of distinctive, easily recognizable, tip clades. These tip clades almost always correspond to a priori species hypotheses, and show nonoverlapping patterns of sequence divergence, making it possible to place species names on a number of immature specimens. Three cases of inconsistency between recovered tip clades and a priori species hypotheses suggest possible introgression between cave-dwelling Cicurina, or alternatively, species synonymy. Although species determination is not possible in these instances, the inconsistencies point to areas of taxonomic ambiguity that require further study. Our molecular phylogenetic sample is largest for the Federally Endangered C. madla. These data suggest that C. madla occurs in more than twice the number of caves as previously reported, and indicate the possible synonymy of C. madla with C. vespera, which is also Federally Endangered. Network analyses reveal considerable genetic divergence and structuring across caves in this species. Although the use of DNA sequences to identify previously 'unidentifiable' specimens illustrates the potential power of molecular data in taxonomy, many other aspects of the same dataset speak to the necessity of a balanced taxonomic approach.     

基于mtDNA COI基因的离腹寡毛实蝇属常见种DNA条形码识别和系统发育分析

【目的】离腹寡毛实蝇属Bactrocera昆虫是最具经济重要性的实蝇类害虫,本研究依据mtDNA COI基因碱基序列对离腹寡毛实蝇属常见实蝇种类进行识别鉴定与系统发育分析。【方法】以口岸经常截获的离腹寡毛实蝇属8个亚属21种实蝇为对象,采用DNA条形码技术,通过对mtDNA COI基因片段 (约650 bp)的测序和比对,以MEGA软件的K2-P双参数模型计算种内及种间遗传距离,以邻接法(NJ) 构建系统发育树。【结果】聚类分析与形态学鉴定结果一致,除11种单一序列实蝇外,其他10种实蝇均各自形成一个单系,节点支持率为99%以上。种内（10种）遗传距离为0.0003~0.0068,平均为0.0043;种间（21种）遗传距离为0.0154~0.2395,平均为0.1540;种间遗传距离为种内遗传距离的35.8倍,而且种内、种间遗传距离没有重叠区域。【结论】基于mtDNA COI基因的DNA条形码技术可以用于离腹寡毛实蝇属昆虫的快速鉴定识别,该技术体系的建立对实蝇类害虫的检测监测具有重要意义。     

Simple identification of mitochondrial lineages in contact zones based on lineage-selective primers

A variety of research projects focus on genetic variation among and within maternal lineages as encompassed by mitochondrial DNA (mtDNA). While mtDNA often differs substantially between species, large differences may also be found within species. The evaluation of such divergent lineages, for example in intraspecific contact zones (hybrid zones), commonly involves sequencing numerous individuals. Large‐scale sequencing is both expensive and labour‐intensive. Based on sequences from 15 individuals, we devised a simple and quick polymerase chain reaction assay for identification of divergent mtDNA lineages in a secondary contact zone of the side‐blotched lizard (Uta stansburiana). The application uses lineage‐selective primers to amplify a lineage‐diagnostic product, and is based on each group of mtDNA haplotypes being a monophyletic assemblage of haplotypes sharing the same maternal ancestry, deeply divergent from the other group. The assay was tested on a larger sample (n = 147) of specimens from the contact zone, confirming its usefulness in quick and reliable identification of mtDNA lineages. This approach can be modified for other species, provided diagnostic lineage variation is available, and may also be performed in simple laboratory settings while conducting fieldwork.     

Multiplex PCR and RFLP approaches for identification of rabbitfish (Siganus) species using mitochondrial gene regions

Molecular assays are described for the identification of six rabbitfish (Siganus) species. A multiplex PCR assay using primers targeting the mitochondrial cytochrome b region simultaneously identifies four species: Siganus canaliculatus, S. fuscescens, S. javus, and S. spinus. Subsequent RFLP assays of multiplex amplicons differentiate between S. virgatus and S. corallinus based on diagnostic fragments from the mitochondrial cytochrome oxidase I region. Assays were validated with known specimens demonstrating accuracy of the molecular identification. Applied to morphologically indistinguishable early developmental stages, these assays can facilitate studies on species-specific spatio-temporal patterns of larval dispersal and population connectivity to aid fishery management.     

Origin of intra-individual variation in PCR-amplified mitochondrial cytochrome oxidase I of Thrips tabaci (Thysanoptera: Thripidae): mitochondrial heteroplasmy or nuclear integration?

The mitochondrial genome is increasingly being used as a species diagnostic marker in insects. Typically, genomic DNA is PCR amplified and then analysed by restriction analyses or sequencing. This analysis system may cause some serious problems for molecular diagnosis. Besides the errors introduced by the PCR process, mtDNA sequence variation of amplified fragments may originate from mtDNA heteroplasmy or from nuclear integrations of mtDNA fragments, both of which have been shown to occur in insects. Here we document abundant variation in PCR-amplified sequences of the mitochondrial cytochrome oxidase I gene of Thrips tabaci. We confirm that the most common haplotype is of mitochondrial origin. Some of the observed mutations were introduced by the amplification process. However, the occurrence of some haplotypes at elevated frequencies indicates that within-individual variation of the respective fragment exists at low levels in T. tabaci. The frequencies of these sequences are too low to negatively affect mtDNA-based molecular diagnosis of T. tabaci. The possible origin of these variant haplotypes is discussed.     

DNA barcoding of African fruit bats (Mammalia, Pteropodidae). The mitochondrial genome does not provide a reliable discrimination between Epomophorus gambianus and Micropteropus pusillus

Sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene have been shown to be useful for species identification in various groups of animals. However, the DNA barcoding approach has never been tested on African fruit bats of the family Pteropodidae (Mammalia, Chiroptera). In this study, the COI gene was sequenced from 120 bats collected in the Central African Republic and belonging to either Epomophorus?gambianus or Micropteropus?pusillus, two species easily diagnosed on the basis of morphological characters, such as body size, skull shape and palatal ridges. Two additional molecular markers were used for comparisons: the complete mitochondrial cytochrome b gene and the intron 7 of the nuclear β-fibrinogen (FGB) gene. Our results reveal an unexpected discordance between mitochondrial and nuclear genes. The nuclear FGB signal agrees with our morphological identifications, as the three alleles detected for E.?gambianus are divergent from the fourteen alleles found for M.?pusillus. By contrast, this taxonomic distinction is not recovered with the analyses of mitochondrial genes, which support rather a polyphyletic pattern for both species. The conflict between molecular markers is explained by multiple mtDNA introgression events from M.?pusillus into E.?gambianus or, alternatively, by incomplete lineage sorting of mtDNA haplotypes associated with positive selection on FGB alleles of M.?pusillus. Our work shows the failure of DNA barcoding to discriminate between two morphologically distinct fruit bat species and highlights the importance of using both mitochondrial and nuclear markers for taxonomic identification.     

基于种特异性COI标记的新入侵种甘蓝粉虱快速鉴定技术

【目的】针对粉虱类害虫种类多、体型微小、形态相似、难以准确快速识别的问题,以新入侵我国大陆的甘蓝粉虱 Aleyrodes proletella (L.)为靶标,以田间常见的其他10种/隐种粉虱为参照,采用基于线粒体DNA细胞色素C氧化酶亚基I (mitochondrial DNA cytochrome c oxidase subunit I, mtDNA COI) 基因的种特异性 (species-specific COI, SS-COI) PCR方法,研究其快速分子检测技术。【方法】利用mtDNA COI基因通用型引物LCO-1490/HCO-2198获得甘蓝粉虱及其他常见粉虱的COI序列,根据测序结果设计种特异性SS-COI引物1对（APZYJF/APZYJR）,其扩增片段大小为384 bp,同时对该对引物的种特异性及灵敏性进行检测。【结果】种特异性检验结果显示,该对引物仅对甘蓝粉虱的mtDNA COI基因具有扩增效果,对我国常见的其他种类的粉虱包括温室粉虱 Trialeurodes vaporariorum (Westwood)、柑橘粉虱 Dialeurodes citri (Ashmead)、螺旋粉虱 Aleurodicus disperses (Russell)、双钩巢粉虱 Paraleyrodes pseudonaranjae Martin、非洲伯粉虱 Bemisia afer (Priesner et Hosny)以及烟粉虱B. tabaci (Gennadius)5个隐种（MED, Asia I, Asia II 1, Asia II 3和China 1）等不具有交叉反应扩增能力。灵敏性检验结果显示,该对引物不仅对不同性别的成虫具有良好的扩增效能,对2-4龄若虫甚至单粒卵或单头初孵若虫亦具有同样的扩增能力,其最低检测阈值为14.00±0.37 pg/μL（相当于1/25 600头雌性成虫）。【结论】该技术体系完全可用于甘蓝粉虱的快速准确识别及检测监测,对有效阻截其进一步扩张蔓延意义重大。     

Organization of mitochondrial DNA in normal and texas male sterile cytoplasms of maize

Recombinant DNA and hybridization techniques have been used to compare the organization of mitochondrial DNA (mtDNA) from normal (N) and Texas male sterile (T) cytoplasms of maize. Bam H1 restriction fragments of normal mtDNA were cloned and used in molecular hybridizations against Southern blots of Bam H1 digested N and T mtDNA. Fifteen of the 35 fragments were conserved in both N and T as indicated by hybridization to comigrating bands in their restriction patterns. Only three fragments produced autoradiographs whose differences could reasonably be attributed to single changes in the cleavage site of the enzyme while approximately half (17/35) of the clones resulted in more complicated differences between N and T. The autoradiographs produced by these 17 clones indicated multiple cleavage site changes and/or sequence rearrangements of the mtDNA. Patterns of six of these 17 clones indicated partial duplication of the sequence and two showed variation in the intensity of hybridization between N and T, which may be related to the molecular heterogeneity phenomenon found in maize mitochondrial genomes. The large proportion of changes observed between N and T mtDNA indicates that rearrangements may have played an important role in the evolution of the maize mitochondrial genome.     

Molecular phylogeny of Megaloceros giganteus--the giant deer or just a giant red deer?

Two fragments of mitochondrial DNA (mtDNA) of the cytochrome b gene (137 bp and 167 bp) were successfully isolated and sequenced from antlers and bones of five specimens of the Giant Deer (Megaloceros giganteus) to examine the phylogenetic position of Megaloceros giganteus within the family Cervidae. This is the first report on ancient DNA (aDNA) sequences from Megaloceros giganteus. A phylogenetic analysis based on parameter-rich models describes the evolutionary relationships between five individuals of fossil Megaloceros giganteus and 37 individuals of 11 extant species of the family Cervidae. The results support a "Cervus-Megaloceros" clade. The phylogenetic positions of sympatric Megaloceros and Cervus elaphus specimens in particular indicate either that the Megaloceros mtDNA gene pool did not evolve for a substantial time period as an entity distinct from Cervus elaphus until its extinction, or that Megaloceros contributed mtDNA to Cervus elaphus or vice versa. The results of this study allow the conclusion that the European Megaloceros giganteus is more related to its modern regional counterparts of the species of Cervus elaphus than recent claims have suggested.     

Mitochondrial DNA rearrangements are correlated with a delayed amplification of the mobile intron (plDNA) in a long-lived mutant of Podospora anserina

A new long-lived mutant of Podospora anserina has been isolated and characterized. Its longevity is maternally inherited as revealed by reciprocal crosses. A molecular analysis resulted in the identification of an amplified DNA species (designated pAL2-1) with homology to mitochondrial DNA (mtDNA). The presence of this DNA species is correlated with mtDNA rearrangements and a delayed amplification of the mobile intron (plDNA).     

Assessing DNA for fish identifications from reference collections: the good,bad and ugly shed light on formalin fixation and sequencing approaches

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.